Our presentations at the Second International Research Conference on HLB in Orlando documented recent progress in characterizing HLB resistance/tolerance as found in Poncirus trifoliata and hybrids of that species with citrus. Some trifoliate hybrid citrus rootstocks, including US-802 and US-897, were demonstrated to become infected by HLB more slowly, to develop smaller amounts of bacterial even after infection, and to exhibit dramatically less symptoms in response to HLB infection than other citrus cultivars. A greenhouse experiment was initiated to compare the utility of the trifoliata-type resistance in the rootstock and scion position in grafted trees. Evidence was presented that documents significantly higher levels of some antimicrobial metabolites in the HLB-resistant trifoliate hybrid germplasm. Gene expression and metabolomic studies are underway to further characterize the genes and metabolites responsible for the resistance that can be used in creation and selection of conventional and transgenic varieties with improved tolerance or resistance to HLB. In this quarter, fruit quality, yield, and/or tree size data were collected from fourteen rootstock and scion field trials. Data was collected from one large cooperative rootstock trial with Ray Ruby grapefruit scion to evaluate rootstock effect on grapefruit quality and sheepnosing. Scion growth was measured on a greenhouse experiment to develop a more rapid way to evaluate CTV resistance and to evaluate supersour rootstocks for tolerance to four different decline strains of CTV. Rooted cuttings of supersour rootstock hybrids were budded with scions to propagate trees for field trials. One hundred supersour-type hybrids were selected from among new progeny for propagation and additional testing. A field trial was planted to compare HLB reaction of standard rootstock varieties to that of new transgenic rootstocks. Budded greenhouse trees for field trials were grown to planting size. Experiments continued to assess the utility of different methods for testing germplasm for resistance or tolerance to Asian Citrus Psyllid (ACP) and HLB disease. Data continued to be collected from four field experiments to assess the HLB tolerance of sweet orange trees on 15 different rootstocks. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). Rootstocks were transformed with an early flowering gene to produce selections that will allow more rapid advancement through generations and thus, more rapid genetic improvement. Selected transgenic rootstocks were challenged with HLB to assess potential resistance, including constructs with two new bacterial resistance genes. Research is continuing to use HLB responsive genes and promoters identified in a previously published gene expression study for engineering resistance in citrus. A microarray analysis was completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. This data is being analyzed now for publication and to guide future breeding and transformation research. The new hybrid rootstock US-942 was officially released for commercial use, based on outstanding performance in several different field trials. Seed of US-942 was provided for distribution to Florida citrus nurseries.
For EDS1 cloning, we currently confirmed by sequencing the cloning of the full-length ctEDS1 and already moved the sequence from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we designed primes to clone the 3′ end sequence of ctSID2, which we were previously unable to obtain with several methods. We performed the RACE reaction and have now obtained this missing sequence. We will subsequently design primers to amplify the full-length sequence of ctSID2. In addition, we did bioinformatics analysis and identified additional 9 citrus homologs of Arabidopsis defense genes that have available sequences in the database. We designed primers for these citrus genes and have been conducting RACE in order to amplify the genes. So far we have obtained the 3′ end sequences for ctNHL1, ctSFD1, and ctFAD7. Further cloning of 5′ end and/or 3′ end sequences of these citrus defense genes are currently underway. We continue to characterize the transgenic plants expressing ctNDR1/pBINplusARS. We obtained 5 homozygous ndr1 + ctNDR1/pBINplusARS. Through HR test and disease resistance assay with infection of the avirulent strain P. syringae avrRpt2, we further confirmed that over-expression of ctNDR1 could complement Arabidopsis ndr1 mutant. We are going to further characterize the defense phenotypes of these transgenic plants.
Over the past quarter, we have continued to develop all aspects of our project. In particular we have progressed in the following areas: 1. Building and testing additional TAL effector and promoter constructs. We have synthetically assembled a number of TAL effector genes matching X. citri TAL effectors and showed that they transcriptionally activate our broad recognition or “super” promoter in a Nicotiana benthamiana system. We have also assembled promoters with individual TAL effector binding sites to test activity and specificity. 2. Testing activation of gene constructs against a diverse world wide collection of X. citri isolates. Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. Additionally, we are preparing and testing X. citri strains with single or multiple disruptions in their TAL effector complement to test the role of specific TAL effector proteins in the disease and resistance process. 3. Stable transformations. The transformed lines generated last Fall and Winter have progressed through selection, shoot formation and rooting, and are now growing in soil. These lines are tested by PCR as they reach adequate size, and positively scored lines have been subjected to pathogen testing by pin-prick assay with X. citri. We have identified several canker resistant transgenic lines. We are currently setting up additional transformations to generate more transgenic material for line testing and with new promoter constructs. 4. Manuscript preparation We are in the process of drafting a manuscript of our results.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but the identification and incorporation of ACP resistance traits from uncultivated Citrus spp. and Citrus relatives is seen as a potential disease management strategy. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field for resistance to natural South Florida populations of ACP. The majority of genotypes surveyed hosted all three life stages of ACP, however there were significant differences among genotypes in the mean ranks for ACP eggs (F = 3.13, df = 86, P < 0.000), nymphs (F = 9.01, df = 86, P < 0.000), and adults (F = 4.21, df = 86, P < 0.000). The only sampled genotype that was completely avoided by all life stages of ACP was Casimiroa edulis, commonly known as white sapote, which was one of the few plants included in the study belonging to the Rutaceae subfamily Toddalioideae. Although not completely avoided, very low levels of ACP were found on two surveyed genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The identification of partial resistance in this species to ACP may prove useful in future citrus breeding programs efforts aimed at controlling the damage caused by reducing the incidence and spread of HLB. In recent psyllid no-choice oviposition studies using six different genotypes of Poncirus trifoliata, with Citrus aurantium and C. macrophylla as susceptible controls, a greatly reduced number of ACP eggs were laid on the Poncirus trifoliata selections when compared with the controls. The post doc assigned to the project resigned in December to take a permanent job elsewhere. A new post doc has been hired and is set to start during late January. A major thrust of upcoming work will be on no-choice experiments with Poncirus trifoiata selections. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae : one with 31 citrus varieties and one with 40 citrus varieties. Both experiments are free-choice studies under greenhouse conditions. A delegation from FAAS will be visiting USDA-ARS during April to coordinate research.
Production of transgenic Citrus plants in the Core Citrus Transformation Facility (CREC) continues to be at the rate of about 100 plants per three months. Orders are being serviced for clients based both in Gainesville and in Lake Alfred. The demand for transgenic material is holding steady. Additional four orders were taken to produce transgenic grapefruit carrying genes harbored in following vectors: pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. However, most of the activities of the facility are directed towards completion of previously placed orders. New orders are being serviced according to the order they were placed. The list of transgenic plants that were delivered within the last quarter includes those concerned with resistance to both bacterial diseases and CTV. Canker and HLB: 1) N1* gene: one Duncan plants; 2) NPR1: three Flame plants and superNPR1-six Flame plants; 3) AS7 gene: two Duncan plants and A13* gene: four Duncan plants; 4) pMKK7 vector: 20 Duncan plants; 5) pMOD1 vector: seven Duncan plants; 6) pNAC1 vector: one Duncan plants; 7) pSuc-NPR1 vector: three Duncan plants. CTV: 1) Gene in p33 vector: 26 Mexican limes, 16 C. macrophylla, and five Hamlin plants. CCTF also produced and delivered eight more Mexican lime plants for the pHK vector order. Change in genotype of these plants is not involved in response to plant pathogens. There are about twenty soil-adapted plants that will be tested by the PCR to confirm the presence of gene of interest in their tissue. Publication supported by this grant: Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266
1- The growth room construction started on October 22nd, 2010, projected finish date is February 11th, 2011. The construction is already one week behind according to the schedule. They are approximately half way done with the wall insulation, the ceiling insulation has not yet begun. We set up a meeting to discuss disposal of the waste stream for the grow room. The director of UF/IFAS Pesticide Information Office, the coordinator of the UF facilities planning and operations and a representative of the EPA were involved in the discussions. A final list of pesticides and chemicals to be used in the grow room was finalized in order to comply with all environmental regulations. 2 – All in vitro clean shoot tips (Hamlin 1-4-1, Valencia 1-14-19 and Pineapple F-60-3) to establish the mother plants were released from Dr. Peggy Sieburth lab, from the Department of Agriculture, Winter Haven. They are still in test tube conditions. The shoot tips are already 3-month old and they are ready for grafting onto rootstocks grown in pots. Since the growth room is not ready, we have transferred them to fresh medium to keep them alive until our growth room is completed. A second transfer of the shoot tips to fresh media is scheduled for January. As mentioned in earlier reports, this material is needed to be grafted on rootstocks in pots at approximately 2 months of growth. Another factor we are worried about is the current rootstock growth conditions. In the lab where they are developing, the lack of appropriate light, temperature, and space to grow them is jeopardizing 6 months worth of work. They are growing but with extreme difficulty. The initial planting material was discarded since it was getting too old and new batches are already growing but until we don’t have a better place where growing them on clean conditions we will continue to struggle. Even if we can occupy the new growth room the last week of January, we are still going to be concerned about achieving our desirable results. 3 – The growth room technician was finally hired and will start working the first week of January. He will go to Spain for training next Spring. 4 – The lab is 80% set up and, after removal of all plants to the growth room, we will clean and finish setting up the lab for full in vitro culture purposes. Supplies and equipment for the growth room will be purchased once it is completed.
Two full genome sequences have been assembled and annotated. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI (US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. This genome will soon be released through the Phytozome portal at JGI, as well as Tree Fruit GDR, and will be presented at the coming International Plant and Animal Genome (PAG) Conference in January 2011. The current version, Citrus clementina 0.90, is based on ~6.4x coverage, and is a very preliminary product released to enable citrus research community access. Additional work in 2011 will vastly improve the assembly through inclusion of BAC end sequences and integration with a high-density genetic linkage map, to yield a chromosome-based assembly with improved annotations. Fifteen BAC clones are being sequenced and assembled to compare with the assembled genome for validation of the assembly. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. This genome sequence is based on ~30x depth of sequence coverage and was assembled using Newbler software; it covers 319 Mb spread over 12,574 scaffolds. Half of the genome is accounted for by 236 scaffolds 251 kb or longer. The current gene set (orange1.1) integrates 3.8 million new ESTs (produced this year) with homology and ab initio-based gene predictions; 25,376 protein-coding loci have been predicted generating a total of 46,147 transcripts. The sweet orange genome also will be presented at PAG in January 2011, and can be accessed through the portals indicated above. The 3.8 million sweet orange ESTs came from 17 different libraries that were produced and sequenced using the 454 platform. They represent various biotic/abiotic challenges including psyllid feeding on young seedlings, canker inoculation, and treatment with salicylic acid, among others. Substantial progress has also been made on the other objectives of this project. Studies comparing the time courses of gene expression in two sets of HLB-inoculated sweet orange and rough lemon plants, representing more susceptible and more tolerant types respectively, have been completed using Affymetrix and Agilent citrus chips (the latter developed by us at UF); some differentially expressed genes have been confirmed by RT-PCR. In addition, comparisons of carbohydrate metabolism and anatomical changes associated with gene expression differences in these same plants have been completed; manuscripts are in submission. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs.
Our research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. During the first year of the grant’s period peptides, proteins and RNAi moieties were tested by feeding them to psyllids using artificial diets. The diet was optimized by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period and we identified suitable buffers and optimal pH. Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from Diaprepes abbreviatus, the citrus root weevil, were found to be excellent candidates; causing high mortalities when fed to psyllids by artifical diet. Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. Three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. In the first quarter of the second year period our studies continued to characterize the cause of increased psyllid mortality induced by feeding of Double-stranded RNA (dsRNA) molecules targeting specific psyllid genes. Large scale experiments were conducted to harvest sufficient RNA for Northern blot characterization of the integrity and abundance of specific psyllid mRNAs that were targeted and showed enhanced insect morality. The Northern blot analyses although cumbersome and time consuming, are essential for complementing Q-RT-PCR based analyses of targeted transcript abundance. To further support and enhance our RNAi research observations using artificial feeding chambers, we developed a detached leaf assay that supports adult and nymph psyllid survival and allows dsRNA uptake into intact citrus leaves on which the psyllid are naturally feeding. Initial results suggest that transcript specific mortality induced by feeding dsRNA to psyllids in artificial diets can be reproduced using the detached citrus leaf assay. The assay was developed to show that low doses of dsRNA circulating within the phloem can shut down key biological genes in psyllids when ingested, and thus support the possibility that RNAi strategies can be developed to control psyllid feeding on citrus and, therefore, control the spread of HLB. As part of this research a dsRNA virus was also discovered in psyllids and was characterized. This virus is present in natural psyllid populations within Florida, but accumulates to higher levels when the psyllids are maintained in greenhouse colonies. Because it is possible that dsRNA viruses can suppress the RNAi machinery of an insect, we are currently developing dsRNA of virus free psyllid colonies to support future RNAi research in psyllids.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. These include Polyamines such as putrecine, spermine and spermidine; and Antioxidants such as lipoic acid, glycine betaine and glutathion. Lipoic acid continues to yield the best results. A carrot suspension culture overlay procedure is also being evaluated. Experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency are also underway, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publication from work on alternative transformation systems: Dutt, M. and J.W. Grosser. 2010. An embryogenic suspension cell culture system for Agrobacterium mediated transformation of citrus. Plant Cell Reports. 29(11): 1251-1260. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ A field trial was established in collaboration with Mr. Orie Lee to evaluate sweet orange seedlings from six selected somaclones of precocious ‘Vernia’ sweet orange under commercial conditions. Juvenile Vernia trees are less thorny than other commercial sweet oranges, and our plan is to girdle the trees to induce early flowering and fruiting once the trees reach adequate size. The goal is to quickly establish a producing grove from juvenile budwood – as necessary to have a system for comparable transgenics. Significant progress was also made to identify rootstocks to enhance early production from juvenile scions, including subsequent transgenics. The 2.5 year old field trial using a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks is showing significant rootstock affects on precocious bearing – the best selections from this trial will be tested with juvenile transgenics, based on yield and fruit quality data to be taken in February. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious ‘Vernia’ sweet orange (including somaclones) were regenerated and successfully micrografted for further study of early flowering and transgene expression. 31 transgenic ‘Vernia’ trees were produced containing four different gene constructs. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
In cloning the three SA genes, EDS1, SID2, and WIN3, we currently confirmed the cloning of the full-length ctEDS1 and are in the process of moving the sequence to the binary vector for plant transformation. We showed in the last progress report that we obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2. In order to amplify the other ends of the two genes, we tried to design different primers for RT-PCR. We also performed TAIL PCR, in which we used citrus genomic DNA as a template in a series of PCR in order to obtain the missing regions of the two genes. However, these attempts were unsuccessful. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we have been doing bioinformatics analysis and have identified additional SA genes that have citrus homologs with available sequence. We are currently design primers to further amplify these additional SA genes. For ctEDS5/pBINplusARS transformation, we obtained 5 Col-0 and 5 eds5-1 carrying the transgene. We are in the process of screening T0 seeds for additional independent transformants. In the meantime, we planted these 10 transgenic plants for disease resistance assay with Pseudomonas infection. We continue to characterize the transgenic plants overexpressing ctNDR1/pBINplusARS, ctNPR1/pBINplusARS, or ctPAD4/pBINplusARS. We obtained 4 homozygous ndr1 + ctNDR1/pBINplusARS and performed disease resistance assay. The recent data confirmed our earlier report that ctNDR1 complemented Arabidopsis ndr1 mutant. Additional analysis will be conducted to verify this result and to further characterize the defense phenotypes of the transgenic plants. For plants overexpressing ctNPR1/pBINplusARS or ctPAD4/pBINplusARS, we did not observe complementation of npr1 or pad4 mutant with transgenic plants currently obtained. We reason that overexpression of these two genes may be toxic or citrus cDNA clones may not be well expressed in Arabidopsis. We are currently trying to clone the genomic fragments of these two genes. We will repeat npr1 or pad4 complementation once we obtain the genomic clones.
During the 2nd quarter of funding, the Core Citrus Transformation Facility (CREC) continued it’s mission of producing transgenic Citrus plants according to the orders from multiple clients. The demand for genetically transformed citrus plants remains high. Most recently, CCTF received three new orders to produce transgenic grapefruit carrying genes harbored in following vectors: p19-5; p20-7; and p21-1. The bulk of the work presently revolves around orders placed in the previous quarter but work also goes on to complete older orders. Out of presently serviced orders, all except two are concerned with resistance of different citrus cultivars to diseases, primarily HLB and canker. The following transgenic citrus plants were delivered to various researchers: Resistance to bacterial diseases-canker and HLB: 1) N1* gene: two Duncan plants; 2) pCIT108P3 vector: two Flame plants; 3) NPR1: three Flame plants and superNPR1-four Hamlin plants; 4) AS7 gene: eight Duncan plants and A13* gene: four Duncan plants; 5) pMOG800 vector: two Duncan plants. Resistance to CTV: 1) Gene in p33 vector: 18 Mexican limes, 16 C. macrophylla, and seven Hamlin plants. Orders not associated with citrus disease resistance: 1) CL1 gene: one Duncan. 2) pHK vector: 12 Mexican limes. During this quarter, more than forty recovered new transgenic plants were soil-adapted, and are ready for PCR testing to confirm the presence of the trasngene of interest. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, evaluated the mechanisms involved in the resistance of kumquat to the citrus canker. MK and Nagami NK developed a hypersensitive response (HR), with necrotic lesions with population of Xanthomonas citri subsp. citri (Xcc) < 5 log units after 168 h in detached leaf and attached leaf assays. Early expression of genes related to programmed cell death associated with HR were identified in MK and NK. The resistance in kumquats has several characteristics associated with HR: 1) Rapid necrosis of leaf tissue in 48-72 h post inoculation in vitro or 72-96h in planta; 2) Disruption of epidermal and mesophyll cells by 72 h; 3) Xcc bacterial ingress limited to few cell layers below the epidermis; 4) Xcc population growth arrested at 72 h coincident with the cellular disruption; 4) Light microscopy and TEM, show death of the cells adjacent to the inoculation site with very few bacteria proliferating; 6) 5) HR-related genes and other putative resistance-related genes expressed early in resistant KN but not in susceptible DG. Behavior of susceptible DG and RL was: 1) No symptoms are detect in susceptible until 72 hr after inoculation and water soaked developes at 168 hr; 2) pustular callus-like lesions erupted through the cuticle by 10-16 days post inoculation (dpi); 4) Xcc populations reach 6 log cfu of Xcc per inoculation site at 168 hr; 5) Xcc population increases up to 15 dpi. Objective 2: Validate the inheritance or resistance for cybrids with susceptible Red grapefruit (RG) and RL with Valencia orange (VO). The putative RL cybrid has been recently been fully analyzed and determined using Single stranded repeat (SSR) analysis to be a cybrid from a mislabeled callus line of Valencia orange and not Meiwa kumquat. Hence the inheritance of resistance is not the HR type but is one of moderate susceptibility compared to high susceptibility in RG. Evidently, there is a definite expression of resistance in the cybrid inherited as a result of presence of the heterologous mitochondrial or chloroplast genome from the VO callus line. Evidence for this is as follows: Intermediate lesion symptoms are observed for RL+VO cybrid in vitro and in-planta. In contrast to development of callus, the inoculated area develops necrosis by 10 dpi. Xcc population plateaus by 10 dpi below the bacteria populations susceptible RL or Red GF. Two types of lesion were observed: necrotic and also callus, suggesting that cell death occurs and arrests the proliferation of Xcc. Expression of HR-related genes is intermediate between MK and RL, further substantiating that some yet to be determined elements of resistance have been inherited in the cybrids. Finally, the current set of Ruby red grapefruit cybrids with VO planted in canker-affected locations on the east coast continue to be more resistant than Red grapefruit trees around them. To expedite and standardize the evaluation of resistance in citrus germplasm, a prototype needle-free device was designed and evaluated for delivery of Xcc into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomates by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results for the hand-held syringe method.
This is a 3-year project with 2 specific aims: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the pBI1.4T-AtMKK7 construct has been mobilized into the Agrobacterium strain EHA105. The culture of the Agrobacterium was used for co-incubation with ‘Duncan’ grapefruit explants. About 1700 explants were incubated and 17 shoots were tested with PCR. Nine of these shoots were positive in the initial screen and all of them were grafted onto Carrizo. The transgenic plants are growing. Further confirmation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line will be performed. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when the transgenic plants are available. For objective 2, we previously did irradiation for the first batch of ‘Duncan’ grapefruit hypocotyl cuttings with a irradiation dosage of 40G. The irradiated cuttings generated significantly fewer shoots than the control, suggesting that the irradiation caused severe damage to the hypocotyl cuttings. Calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control have been transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We prepared another batch of explants (90 tubes of ‘Duncan’ grapefruit seedlings) for irradiation. A total of 90 plates of hypocotyl cuttings (each plate with 40-50 stem pieces) was irradiated with a dosage of 30G on 06/28/10. The irradiated stem pieces were placed on non-selective shooting medium. Ten plates of hypocotyl cuttings were kept as non-irradiated controls, for future comparison. The plates are kept under 14 hour photoperiod. Shoots generated from the irradiated hypocotyls were transferred onto selective medium with 0.2 mM of sodium iodoacetate. We are also preparing the third batch of hypocotyls for irradiation.
About 500 supersour-type (SS) rootstock hybrids have been selected for propagation and further testing. Selected SS rootstocks are being evaluated for tolerance to CTV quick decline and propagated for placement into field trials. Commercial cooperators are being identified who will host early stage trials of some SS rootstocks. Rootstock liners were budded with scions to prepare trees for trials. Budded greenhouse trees for field trials were grown to planting size. A new field trial was planted to assess the interaction of rootstocks and scion cultivars on tree performance under an open hydroponic management system. Data on tree size and HLB titer were collected from several rootstocks trials to assess rootstock effect on tree growth under HLB disease pressure. Data were collected from a trial planted on trellis to examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. This information will be valuable to accelerate the pace of development for new rootstocks and scions. Studies continue to assess citrus germplasm tolerance to Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. In a new greenhouse study, Poncirus trifoliata, Cleopatra mandarin, and several hybrid selections were inoculated with HLB to further evaluate the apparent HLB tolerance in some trifoliata-type selections revealed in a previously completed greenhouse study. Greenhouse trees inoculated with Citrus tristeza virus (CTV) were tested for virus titer in preparation for CTV-induced decline evaluation of supersour rootstocks. More than fifty citrus genotypes and citrus relatives, as well as thousands of progeny from crosses, have been challenged by natural inoculation with Liberibacter in the field, and data are being collected on HLB symptoms and Liberibacter titer by PCR. Detailed information is being collected on HLB tolerance and tree performance in four rootstock field trials. All citrus germplasm and cultivars become infected with Liberibacter when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Preliminary evaluations were completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Efforts continue to transform trees with seven different promoters and three new anti-bacterial genes targeted at producing HLB-resistant cultivars. Testing of transgenic plants for HLB resistance continues under the resources provided by this grant. Genetic transformation was used to introduce the citrus FT gene for induction of early flowering into citrus scion and rootstock germplasm. Commercial field plantings of the new seedless mandarin cultivar ‘Early Pride’ were planned with cooperators. The final revision of the release notice for US-942 rootstock was prepared and submitted for official approval.
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